Method for analyzing samples of a biological fluid

Abstract

The present invention relates to methods for continuous or near-continuous separation and purification of samples, particularly biological samples, to reduce the number or volume of non-targeted analytes in those samples to enable improved mass spectrometric analysis of analytes of interest, and apparatuses for conducting those methods, utilizing a transport agent with a core domain and a binding domain, the transport agent exceeding 200 kiloDaltons or 10 nm, and the binding domain targeted to the analytes of interest, in conjunction with size-exclusion based chromatography to separate the transport agent-analyte of interest complex from non-targeted analytes that are not bound, or are only non-specifically bound, to the transport agent.

What is claimed is: 
     
       1. A method of qualitatively and quantitatively analyzing samples of biological fluid, said method comprising the steps of:
 (a) providing a sample for analysis; 
 (b) selecting at least one ATP, each said ATP comprising a core domain and a binding domain; 
 (c) adding a quantity of said at least one ATP to said sample to create a sample mixture; 
 (d) providing conditions suitable for analytes of interest present in said sample to bind to said at least one ATP; 
 (e) loading said sample mixture into an apparatus for analysis, said apparatus comprising at least one stripping column, at least one disassociating portion fluidly connected to said stripping column, and at least one enriching column fluidly connected to said disassociating portion; 
 (f) moving said sample mixture through said at least one stripping column at a first rate; 
 (g) moving said sample mixture through said disassociating portion at a second rate selected to allow substantially all of said analytes of interest to disassociate from said at least one ATP; and 
 (h) moving said sample mixture through said enriching column at a third rate selected to allow chromatographic separation of analytes of interest; 
 (i) collecting elution from said enriching column; and 
 (j) analyzing elution from said enriching column to determine the presence or relative amount of analytes of interest;
 wherein said stripping column comprises a container, a first stationary phase, and a first mobile phase, said first stationary phase comprises at least one size-restricted access sorbent, said size-restricted access sorbent comprises an inactive external portion, an active internal portion, and pores providing access from said external portion to said internal portion, said pores having a diameter smaller than the diameter of said ATPs, and wherein said first mobile phase comprises a solvent such that said sample mixture moves through said stripping column within said first mobile phase; 
 wherein said disassociating portion comprises one or more containers and said disassociating portion environment is adapted to conditions in which analytes of interest will substantially disassociate from said at least one ATP; and 
 wherein said enriching column comprises a second stationary phase and a second mobile phase, said second stationary phase comprises a least one sorbent selected to induce chromatographic separation in analytes of interest and said second mobile phase comprises a solvent, and said enriching column environment is adapted to conditions in which said analystes of interest will be substantially prevented from rebinding to said at least one ATP. 
 
 
     
     
       2. The method of  claim 1 , wherein said ATP comprises at least one of an organic macromolecule with an atomic mass of at least 500 kilodaltons, silica, Ficoll with atomic weight of at least 400 kiloDaltons, dextrans with atomic weight of at least 1000 kiloDaltons, and an organic polymer. 
     
     
       3. The method of  claim 2 , wherein each said ATP has a diameter in the range of 10 nm to 200 nm. 
     
     
       4. The method of  claim 3 , wherein said size-restricted access sorbent comprises an external portion, an internal portion, and pores separating said external portion from said internal portion and said pores each have a diameter of approximately 30 nm. 
     
     
       5. The method of  claim 4 , wherein said disassociating portion comprises a capillary tube having volume in the range of 1 uL to 10 uL. 
     
     
       6. The method of  claim 5 , wherein the cross-sectional area of said first mobile phase is equal to or less than approximately 3×10 7  um 2 . 
     
     
       7. The method of  claim 6 , wherein said binding domain comprises at least one of an antibody, avidin, a lectin, protein A, protein G, and an aptamer.